Irxl1 mutant mice show reduced tendon differentiation and no patterning defects in musculoskeletal system development

Irxl1 (Iroquois-related homeobox like-1) is a newly identified three amino-acid loop extension (TALE) homeobox gene, which is expressed in various mesoderm-derived tissues, particularly in the progenitors of the musculoskeletal system. To analyze the roles of Irxl1 during embryonic development, we generated mice carrying a null allele of Irxl1. Mice homozygous for the targeted allele were viable, fertile, and showed reduced tendon differentiation. Skeletal morphology and skeletal muscle weight in Irxl1-knockout mice appeared normal. Expression patterns of several marker genes for cartilage, tendon, and muscle progenitors in homozygous mutant embryos were unchanged. These results suggest that Irxl1 is required for the tendon differentiation but dispensable for the patterning of the musculoskeletal system in development.     

Skeletal muscle-specific expression of PGC-1α-b, an exercise-responsive isoform, increases exercise capacity and peak oxygen uptake

Background

Maximal oxygen uptake (VO_2max) predicts mortality and is associated with endurance performance. Trained subjects have a high VO_2max due to a high cardiac output and high metabolic capacity of skeletal muscles. Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α), a nuclear receptor coactivator, promotes mitochondrial biogenesis, a fiber-type switch to oxidative fibers, and angiogenesis in skeletal muscle. Because exercise training increases PGC-1α in skeletal muscle, PGC-1α-mediated changes may contribute to the improvement of exercise capacity and VO_2max. There are three isoforms of PGC-1α mRNA. PGC-1α-b protein, whose amino terminus is different from PGC-1α-a protein, is a predominant PGC-1α isoform in response to exercise. We investigated whether alterations of skeletal muscle metabolism by overexpression of PGC-1α-b in skeletal muscle, but not heart, would increase VO_2max and exercise capacity.

Methodology/Principal Findings

Transgenic mice showed overexpression of PGC-1α-b protein in skeletal muscle but not in heart. Overexpression of PGC-1α-b promoted mitochondrial biogenesis 4-fold, increased the expression of fatty acid transporters, enhanced angiogenesis in skeletal muscle 1.4 to 2.7-fold, and promoted exercise capacity (expressed by maximum speed) by 35% and peak oxygen uptake by 20%. Across a broad range of either the absolute exercise intensity, or the same relative exercise intensities, lipid oxidation was always higher in the transgenic mice than wild-type littermates, suggesting that lipid is the predominant fuel source for exercise in the transgenic mice. However, muscle glycogen usage during exercise was absent in the transgenic mice.

Conclusions/Significance

Increased mitochondrial biogenesis, capillaries, and fatty acid transporters in skeletal muscles may contribute to improved exercise capacity via an increase in fatty acid utilization. Increases in PGC-1α-b protein or function might be a useful strategy for sedentary subjects to perform exercise efficiently, which would lead to prevention of life-style related diseases and increased lifespan.     

Reduced rate of neural differentiation in the dentate gyrus of adult dysbindin null (sandy) mouse

Genetic variations in the gene encoding dysbindin has consistently been associated with schizophrenia and bipolar disorder, although little is known about the neural functions carried out by dysbindin. To gain some insight into this area, we took advantage of the readily available dysbindin-null mouse sandy (sdy-/-) and studied hippocampal neurogenesis using thymidine analogue bromodeoxuridine (BrdU). No significant differences were found in the proliferation (4 hours) or survival (1, 4 and 8 weeks after the last BrdU injection) of progenitors in the subgranular regions of the dentate gyrus between sdy-/- and sdy+/+ (control) mice. However, 4 weeks after the last BrdU injection, a significant reduction was observed in the ratio of neuronal differentiation in sdy-/- when compared to that of sdy+/+ (sdy+/+ = 87.0 ± 5.3% vs. sdy-/- = 71.3 ± 8.3%, p = 0.01). These findings suggest that dysbindin plays a role during differentiation process in the adult hippocampal neurogenesis and that its deficit may negatively affect neurogenesis-related functions such as cognition and mood.     

Impact on malaria parasite multiplication rates in infected volunteers of the protein-in-adjuvant vaccine AMA1-C1/Alhydrogel+CPG 7909

Background

Inhibition of parasite growth is a major objective of blood-stage malaria vaccines. The in vitro assay of parasite growth inhibitory activity (GIA) is widely used as a surrogate marker for malaria vaccine efficacy in the down-selection of candidate blood-stage vaccines. Here we report the first study to examine the relationship between in vivo Plasmodium falciparum growth rates and in vitro GIA in humans experimentally infected with blood-stage malaria.

Methods

In this phase I/IIa open-label clinical trial five healthy malaria-naive volunteers were immunised with AMA1/C1-Alhydrogel+CPG 7909, and together with three unvaccinated controls were challenged by intravenous inoculation of P. falciparum infected erythrocytes.

Results

A significant correlation was observed between parasite multiplication rate in 48 hours (PMR) and both vaccine-induced growth-inhibitory activity (Pearson r = −0.93 [95% CI: −1.0, −0.27] P = 0.02) and AMA1 antibody titres in the vaccine group (Pearson r = −0.93 [95% CI: −0.99, −0.25] P = 0.02). However immunisation failed to reduce overall mean PMR in the vaccine group in comparison to the controls (vaccinee 16 fold [95% CI: 12, 22], control 17 fold [CI: 0, 65] P = 0.70). Therefore no impact on pre-patent period was observed (vaccine group median 8.5 days [range 7.5–9], control group median 9 days [range 7–9]).

Conclusions

Despite the first observation in human experimental malaria infection of a significant association between vaccine-induced in vitro growth inhibitory activity and in vivo parasite multiplication rate, this did not translate into any observable clinically relevant vaccine effect in this small group of volunteers.

Trial Registration

ClinicalTrials.gov [{"type":"clinical-trial","attrs":{"text":"NCT00984763","term_id":"NCT00984763"}}NCT00984763]     

Tolerance of spermatogonia to oxidative stress is due to high levels of Zn and Cu/Zn superoxide dismutase

Background

Spermatogonia are highly tolerant to reactive oxygen species (ROS) attack while advanced-stage germ cells such as spermatozoa are much more susceptible, but the precise reason for this variation in ROS tolerance remains unknown.

Methodology/Principal Findings

Using the Japanese eel testicular culture system that enables a complete spermatogenesis in vitro, we report that advanced-stage germ cells undergo intense apoptosis and exhibit strong signal for 8-hydroxy-2′-deoxyguanosine, an oxidative DNA damage marker, upon exposure to hypoxanthine-generated ROS while spermatogonia remain unaltered. Activity assay of antioxidant enzyme, superoxide dismutase (SOD) and Western blot analysis using an anti-Copper/Zinc (Cu/Zn) SOD antibody showed a high SOD activity and Cu/Zn SOD protein concentration during early spermatogenesis. Immunohistochemistry showed a strong expression for Cu/Zn SOD in spermatogonia but weak expression in advanced-stage germ cells. Zn deficiency reduced activity of the recombinant eel Cu/Zn SOD protein. Cu/Zn SOD siRNA decreased Cu/Zn SOD expression in spermatogonia and led to increased oxidative damage.

Conclusions/Significance

These data indicate that the presence of high levels of Cu/Zn SOD and Zn render spermatogonia resistant to ROS, and consequently protected from oxidative stress. These findings provide the biochemical basis for the high tolerance of spermatogonia to oxidative stress.     

Downregulation of adipose triglyceride lipase in the heart aggravates diabetic cardiomyopathy in db/db mice

Adipose triglyceride lipase (ATGL) was recently identified as a rate-limiting triglyceride (TG) lipase and its activity is stimulated by comparative gene identification-58 (CGI-58). Mutations in the ATGL or CGI-58 genes are associated with neutral lipid storage diseases characterized by the accumulation of TG in multiple tissues. The cardiac phenotype, known as triglyceride deposit cardiomyovasculopathy, is characterized by TG accumulation in coronary atherosclerotic lesions and in the myocardium. Recent reports showed that myocardial TG accumulation is significantly higher in patients with diabetes and is associated with impaired left ventricular diastolic function. Therefore, we investigated the roles of ATGL and CGI-58 in the development of myocardial steatosis in the diabetic state. Histological examination with oil red O staining showed marked lipid deposition in the hearts of diabetic fatty db/db mice. Cardiac triglyceride and diglyceride contents were greater in db/db mice than in db/+ control mice. Next, we determined the expression of genes and proteins that affect lipid metabolism, and found that ATGL and CGI-58 expression levels were decreased in the hearts of db/db mice. We also found increased expression of genes regulating triglyceride synthesis (sterol regulatory element-binding protein 1c, monoacylglycerol acyltransferases, and diacylglycerol acyltransferases) in db/db mice. Regarding key modulators of apoptosis, PKC activity, and oxidative stress, we found that Bcl-2 levels were lower and that phosphorylated PKC and 8-hydroxy-2′-deoxyguanosine levels were higher in db/db hearts. These results suggest that reduced ATGL and CGI-58 expression and increased TG synthesis may exacerbate myocardial steatosis and oxidative stress, thereby promoting cardiac apoptosis in diabetic mice.     

Neonicotinoid resistance and cDNA sequences of nicotinic acetylcholine receptor subunits of the western flower thrips Frankliniella occidentalis (Thysanoptera: Thripidae)

The western flower thrips, Frankliniella occidentalis (Pergande), is difficult to control because of high insecticide resistance. In this study, susceptibility to major insecticides was examined in two Japanese strains (H-1 and KC) and a Chinese strain (BJ) using a leaf-dipping method. All three strains were resistant to permethrin and acetamiprid at agriculturally recommended doses. The median lethal concentration (LC₅₀) for acetamiprid was 1720 ppm in strain H-1, 4780 ppm in strain KC and >6680 ppm in strain BJ. In the presence of piperonyl butoxide, an inhibitor of cytochrome P450 monooxygenases, the LC₅₀ for acetamiprid was 312 ppm in strain H-1, 837 ppm in strain KC and 1250 ppm in strain BJ. These results suggested that metabolism by cytochrome P450 monooxygenases is involved in acetamiprid resistance in these strains, though other factors also seem to play a role. Furthermore, cDNA cloning of the nicotinic acetylcholine receptor (nAChR) subunits was performed using degenerate primers, and the presence or absence of a point mutation in nAChR β1 was confirmed. The R81T mutation that had been reported in Myzus persicae (Sulzer) nAChR β1 was not found in F. occidentalis strains tested in this study.     

Resveratrol Improves Cardiomyopathy in Dystrophin-deficient Mice through SIRT1 Protein-mediated Modulation of p300 Protein

Cardiomyopathy is the main cause of death in Duchenne muscular dystrophy. Here, we show that oral administration of resveratrol, which leads to activation of an NAD⁺-dependent protein deacetylase SIRT1, suppresses cardiac hypertrophy and fibrosis and restores cardiac diastolic function in dystrophin-deficient mdx mice. The pro-hypertrophic co-activator p300 protein but not p300 mRNA was up-regulated in the mdx heart, and resveratrol administration down-regulated the p300 protein level. In cultured cardiomyocytes, cardiomyocyte hypertrophy induced by the α₁-agonist phenylephrine was inhibited by the overexpression of SIRT1 as well as resveratrol, both of which down-regulated p300 protein levels but not p300 mRNA levels. In addition, activation of atrial natriuretic peptide promoter by p300 was inhibited by SIRT1. We found that SIRT1 induced p300 down-regulation via the ubiquitin-proteasome pathway by deacetylation of lysine residues for ubiquitination. These findings indicate the pathological significance of p300 up-regulation in the dystrophic heart and indicate that SIRT1 activation has therapeutic potential for dystrophic cardiomyopathy.     

Mutant firefly luciferases with improved specific activity and dATP discrimination constructed by yeast cell surface engineering

Pyrosequencing system utilizing luciferase is one of the next-generation DNA sequencing systems. However, there is a crucial problem with the current pyrosequencing system: luciferase cannot discriminate between ATP and dATP completely, and dATPαS must be used as the dATP analogue. dATPαS is expensive and has low activity for the enzyme. If luciferase can clearly recognize the difference between ATP and dATP, dATP could be used instead of the expensive dATPαS in the pyrosequencing system. We attempted to prepare a novel luciferase with improved specific activity and dATP discrimination with the molecular display method. First, we selected two amino acid residues, Ser440 and Ser456, as target residues for mutation from the whole sequence of Photinus pyralis luciferase; we comprehensively mutated these two amino acids. A mutant luciferase library was constructed using yeast cell surface engineering. Through three step-wide screenings with individual conditions, we easily and speedily isolated three candidate mutants from 1,152 candidates and analyzed the properties of these mutants. Consequently, we succeeded in obtaining interesting mutant luciferases with improved specific activity and dATP discrimination more conveniently than with other methods.